Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and sub sequently into internucleosomal fragments. DNA fragmentation factor (DFF) i s implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF 40 subunits, the former of which acts as a chaperone and inhibitor of the c atalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-med iated DNA fragmentation in apoptosis was investigated in primary fibroblast s from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into similar to 50-kb fragments in DFF45( +/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP -ribose) polymerase (PARP), which requires PARP binding to DNA strand break s, and the consequent depletion of the PARP substrate NAD were markedly del ayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic si gnaling, including the loss of mitochondrial membrane potential and the rel ease of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) f ibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the D NA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochro me c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitoc hondrial dysfunction, and caspase-3 activation, contributes to an amplifica tion loop in the death process.